The invention relates to methods and compounds for inhibiting or stimulating presenilin 1 and related pharmaceuticals and diagnostic agents.
Since the discovery of the presenilins in 1995, a significant effort has been made to understand their function and to associate them with a well-defined molecular scheme, especially that involved in programmed cell death. All of this suggests that the presenilins are xe2x80x9cgenes for life and deathxe2x80x9d. PS1 and PS2 are integral membrane proteins with 6 to 9 transmembrane domains situated in the endoplasmic reticulum and the early Golgi complex. PS1 is strongly homologous to PS2.
It has recently been demonstrated that the genes of presenilin 1 and 2 were involved in the molecular phenomena at the root of familial Alzheimer""s disease. Non-sense mutations in the presenilin 1 gene (PS1) have been found in the most aggressive form of familial early onset Alzheimer""s disease, especially intervening at an early stage in the disease (Sherrington et al., Nature 375: 754-760 (1995), herein incorporated by reference).
The product of the presenilin 2 gene forms stable complexes with precursor proteins of beta-amyloid. Beta-amyloid is the principle molecule formed in typical plaques appearing in people suffering from Alzheimer""s disease. For PS1, mutant transgenic mice show a high level of the 42 precursor protein of beta-amyloid, there even supplying a functional link with what takes place in Alzheimer""s disease. The intracellular expression of beta-amyloid proteins under a specific promoter of the neurons in transgenic mice leads to neurodegeneration and increased expression of p53 has been observed in some of these lesions. The overexpression of PS2 in the differentiated PC12 cells of nerve growth factor increases apoptosis initiated by the elimination of trophic factors. In addition, mutations in PS2 could induce apoptosis, even with elimination of trophic factors. During development, anomalies of the skeletons and of the central nervous system appear in presenilin 1-deficient mice. In addition, PS1 is necessary for the expression of Notch 1 and of DLL1 in the development of the paraxial mesoderm. The presenilin proteins seem to show a complex alternative cleavage during apoptosis. PS1 seems to be an active member of the molecular pathways p53-p21 Waf1 of apoptosis and of tumoral suppresion and it is important to recall that expression of intact p53 is necessary to ensure the complex functions of the central nervous system.
To this date, a series of cDNA molecules regulated by p53 during apoptosis and tumoral suppresion have been identified in WO-A-97/22695, which is herein incorporated by reference. The cDNAs were cloned by differential analysis of the mRNAs. TSIP2 (Tumour Suppressor Inhibited Pathway Clone 2) was cloned in a fragment of 90 bp. Used as a probe, the cDNA of TSIP2 revealed two bands by means of Northern blot analysis, one a strong one of 3 kb and the other a weaker one of 7 kb. These mRNAs are inhibited after expression of the wild-type p53 function. This model system takes advantage of the val-135 mutant of p53, which is sensitive to temperature, stably transfected in M1 myeloid cells. In one of these transfectants, exemplified by the LTR 6 cells, 37 to 32xc2x0 C. passage induces the expression of a functional wild-type p53 resulting in an apoptosis phenomenon. The cDNA fragment of TSIP2 initially of 90 bp cloned at the start did not have any homology with any sequence known in the data bases. This fragment was extended to the whole of the cDNA using an RACE-PCR. After sequencing of an additional fragment of 700 bp, it turned out that TSIP2 was identical to the PS1 gene of mice. The inventors have discovered that the inhibition of the expression of the mRNA of PS1 was confirmed by a Northern blot analysis using the cDNA probe in its entirety in the M1-LTR6 model. Regulation intervenes very rapidly after passage at 32xc2x0 C. and is indubitable after induction of wild-type p53 for two hours. This indicates that the modulation of the expression of PS1 intervenes at an early stage of the cell death programme, when no cell has yet died of apoptosis.
The present invention is based on the discovery that inhibition of PS1 by p53 or p21 or a molecule, such as an antisense molecule, induces apoptosis (cell death). Accordingly, it is possible to treat diseases associated with excessive cell growth, such as cancer, by inhibiting PSI expression. Another aspect of the invention is based on the discovery that wild type PSI inhibits p53 induced apoptosis. Accordingly, it is possible to treat diseases wherein it is desirable to inhibit cell death by stimulating the PS1 gene or inhibiting p53 or p21.
Thus, one embodiment of the invention relates to a compound that inhibits the cellular expression of a nucleotide sequence corresponding to all or part of the presenilin 1 gene, said compound comprising an antisense molecule comprising the antisense of a polynucleotide sequence selected from the group consisting of the entire polynucleotide sequence of the presenilin 1 gene, a fragment of the sequence of the presenilin 1 gene, the promoter or other regulatory sequence of the presenilin 1 gene and a polynucleotide sequence that is at least 90% homologous to the polynucleotide sequence of the presenilin 1 gene. This compound may be an antisense molecule comprising the antisense of the sequence of FIG. 4, or a fragment thereof, preferably a cDNA antisense.
In another embodiment, the invention relates to a compound that binds the product of the presenilin 1 gene comprising an antibody that specifically binds said product.
In another embodiment, the invention relates to a compound for inhibiting the cellular expression of the presenilin 1 gene comprising a molecule that activates p53 or p21.
Another embodiment relates to a method of treating a patient having a condition characterized by excessesive cell growth, comprising administering to a patient a cell growth inhibiting amount of a compound, wherein in an in vitro bioassay said compound inhibits the expression of the presenilin 1 gene.
In another embodiment, the invention relates to a compound that stimulates the cellular expression of a nucleotide sequence corresponding to all or part of the presenilin 1 gene.
In another embodiment, the invention relates to a compound that activates the cellular expression of the presenilin 1 gene by interfering with the metabolic pathway of p53 or p21. This compound may be an antibody that binds p53 or p21.
In another embodiment, the invention relates to a method of inhibiting apoptosis in cells comprising transfecting said cells with an expression vector comprising the polynucleotide sequence of the presenilin 1 gene, a fragment of the sequence of the presenilin 1 gene or a polynucleotide sequence that is at least 90% homologous to the polynucleotide sequence of the presenilin 1 gene.
Another embodiment relates to a pharmaceutical composition comprising an expression vector comprising the polynucleotide sequence of the presenilin 1 gene, a fragment of the sequence of the presenilin 1 gene or a polynucleotide sequence that is at least 90% homologous to the polynucleotide sequence of the presenilin 1 gene.
Another embodiment relates to a method of detecting the presence of the presenilin 1 gene in a mammalian tissue sample, the method comprising the steps of:
(a) contacting said tissue sample with a cDNA fragment of TSIP 2 or with the above desribed molecule that binds the presenilin 1 gene under conditions of hybridization, and
(b) detecting the formation of a hybrid of said molecule with the presenilin 1 gene.
In yet another embodiment, the invention relates to a method of screening for drugs that cause cell death comprising contacting a drug with the presenilin 1 gene and detecting whether the drug inhibits expression of said gene, wherein inhibition of expression indicates the drug""s potential use in causing cell death.